This invention relates to measuring fluorescence and properties derived from fluorescence in materials.
In conventional fluorescence microscopy, a sample, such as a biological specimen is stained with fluorophores before being illuminated by light of a relatively short wavelength. The illumination light, which typically is provided from a laser, excites the fluorophores into a higher energy state where they remain for a short period of time, before returning to their original energy state while emitting fluorescent light of a wavelength longer than the excitation wavelength. In a fluorescence microscope, the emitted fluorescent light is collected by an objective lens of the microscope and is passed through the optical system of the microscope, such that it can be viewed by a user, for example, through the eyepieces of the microscope, or on a display screen of a video system that is connected to the microscope's optical system. In many cases, both the excitation light and the fluorescent light share an optical path through the microscope's optical system, and can be separated as needed, by optical components such as dichroic mirrors that reflect light above the excitation wavelengths while passing the excitation light.
The systems that have found most use in laboratories generally use visible fluorescence of materials and visible light sources. The spatial resolution that can be obtained is determined by the specific optical setup. In some cases, the laboratory experimental setups use pulsed laser light to improve the quality of the fluorescence image. Laboratory arrangements are often used to detect biomolecular reactions and interactions that can be probed by fluorescent methods. Fluorescent dyes are commonly used to examine cells by staining portions of the cells. For more routine imaging analyses, or assays, the excitation light source can illuminate a portion of an object to be examined, such as one microlocation in an array of microlocations.
For reasons of image contrast or signal discrimination, there is often a need to improve the resolution and eliminate background noise in the focal region of the sample that is being studied, as biological samples in particular are fairly transparent and light collection over a too wide depth of focus may obscure the specific details that are being studied of the biological sample. Current solutions to this problem include confocal laser scanning microscopy or wide-field deconvolution technologies, which generate optical “slices” or cross-sections that include only the in-focus information. Another technique is the use of two-photon (2P) excitation produced by an infrared ultra-short, pulsed laser beam. In two-photon systems, the pulsed laser allows the same fluorophores to be excited by photons of twice the wavelength than those used in single photon systems, but the longer wavelength photons are not absorbed by the biological sample, which results in decreased toxicity to living cells and decreased photo bleaching. Furthermore, the infrared wavelength excitation significantly reduces scattering within the tissue as the scattering coefficient is proportional to the inverse fourth power of the excitation wavelength, resulting in penetration deeper into the specimen.
Fluorescent systems of this kind typically work well in laboratory settings. However, in the chemical and biotechnology industry, there is often a need to analyze a large number of samples in a time and cost-efficient manner, and due to the different requirements in these environments, the above configurations are often not suitable or possible to use. Therefore, what is needed is an improved apparatus that can be used to analyze an array of samples or objects in an efficient manner, while having the ability to discriminate against background noise.